Stable biodegradable, high water absorbable polyglutamic acid hydrogel by 3-dimensional cross-linking and its preparation method

ABSTRACT

The present invention relates to a method for the production of a stable biodegradable, high water absorbable γ-polyglutamic acid (γ-PGA) hydrogel by directly cross-linking
         (A) a γ-polyglutamic acid (γ-PGA), a γ-polyglutamate, or a mixture thereof, and optionally a polysaccharide containing a carboxylic and/or carboxylate group, an amino acid, or a mixture thereof; and/or   (B) a microbial culture broth containing a γ-polyglutamic acid (γ-PGA), a γ-polyglutamate, or a mixture thereof, and optionally a polysaccharide containing a carboxylic and/or carboxylate group, an amino acid, or a mixture thereof, with a cross-linker comprising a compound having three or more functional groups or a mixture of a compound having three or more functional groups and a compound having two functional groups,   wherein each of the functional groups can react with a carboxylic group (—COOH), carboxylate group (—COO − ), aldehyde group (—CHO), hydroxyl (—OH), carbonyl group (—CO), sulfone group (—SO 2 ), amino group (—NH 2 ) or nitro group (—NO 2 ), or a mixture thereof.       

     The present invention further relates to a stable biodegradable, high water absorbable γ-polyglutamic acid (γ-PGA) hydrogel and its uses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/740,977, filed on Dec. 19, 2003, the contents of which areincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a stable biodegradable, high waterabsorbable γ-polyglutamic acid (γ-PGA) hydrogel by 3-dimensionalcross-linking, and its preparation method and uses.

BACKGROUND OF THE INVENTION

In recent years, water absorbable hydrogels have been used not only asthe materials for paper diapers and tampons, but also as liquidabsorbents for use in medical care, construction, civil engineering,building, etc. Moreover, the water absorbable hydrogels can also be usedas texture enhancers, fresh-keeping agents for foods, important basalmaterial for greening engineering in the fields of horticulture andother agricultural applications.

Conventional methods for preparing water absorbable hydrogels usestarches and celluloses cross-linked with acrylnitrile to formacrylate-based water absorbable hydrogels. Although such acrylate-basedhydrogels are cheap, they can be partially decomposed by microorganismsin the soil and encounter difficulties in waste treatment and problemsof toxicity toward human bodies. It is believed that imparting waterabsorbable hydrogels with good biodegradability will resolve the problemregarding the waste treatment. Therefore, there is a great demand forbiodegradable, water absorbable hydrogels in view of the increasingenvironmental concerns.

In order to overcome the aforementioned problem, conventional techniquesinvolve using biodegradable starch-based, hyaluronic acid-based, orpolyamino acid-based, or materials as the starting materials for thepreparation of biodegradable, water absorbable hydrogels. The methodsfor the preparation of polyamino acid-based, cross-linked products havebeen disclosed in the prior art, such as JP 6-322358, JP 7-224163, JP7-309943, JP 7-300563, JP 10-298282, and JP 11-343339. For example, JP6-322358 indicates that a solution of γ-PGA can be cross-linked via anelectronic polymerization mechanism by using strong gamma-irradiation,so as to form γ-PGA based, cross-linked product. However, the equipmentsfor producing γ-PGA based, cross-linked products through irradiation isvery complicated and restricted, such that the production procedure isdifficult and inconvenient. JP 11-343339 discloses another method forpreparing cross-linked γ-PGA product, comprising isolating a highconcentration of γ-PGA from a culture broth, and using the isolatedγ-PGA as the starting material for the cross-linking reaction with adi-epoxy compound to obtain a biodegradable, water absorbable hydrogel.Nonetheless, such method not only has the drawback associated with therequirement of a high concentration of γ-PGA, obtained through theprocedures including a cell separation and extraction of γ-PGA from amicrobial culture broth through a refining step, but also requiresparticular operational equipments to improve the solubility of γ-PGA andthe di-epoxy compound in a solvent. Moreover, the above method usesγ-laser irradiation to complete the cross-linking reaction between γ-PGAand the di-epoxy compound. Obviously, the preparation technologydisclosed in JP 11-343339 also causes the problems regarding an increasein cost and inconvenience in preparation procedure.

Moreover, JP 5-301904 discloses polysaccharides produced fromAlcaligenes letus B16. U.S. Pat. No. 4,772,419 also discloses a methodfor the preparation of cross-linked polysaccharide products.

Conventional methods for manufacturing cross-linked γ-PGA productsrequire complicated processing procedures, such as the control andoperation of complicated irradiation equipments and the separation andrefining steps. Also, γ-PGA hydrogel products obtained by knowntechnology are relatively unstable and easily decomposed in few days (3to 5 days) after fully swelling in water at room temperature.Surprisingly, the inventors of the present application have found thatgood biodegradable, high water absorbable γ-PGA hydrogels having up to5,000 times water absorption capacity, improved firmness, and long-termstability after full swelling in water or an aqueous medium can bedirectly, simply, and successfully prepared.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a method for the production of a stablebiodegradable, high water absorbable γ-polyglutamic acid (γ-PGA)hydrogel, by directly cross-linking

(A) a γ-polyglutamic acid (γ-PGA), a γ-polyglutamate, or a mixturethereof, and optionally a polysaccharide containing a carboxylic and/orcarboxylate group, an amino acid, or a mixture thereof; and/or

(B) a microbial culture broth containing a γ-polyglutamic acid (γ-PGA),a γ-polyglutamate, or a mixture thereof, and optionally a polysaccharidecontaining a carboxylic and/or carboxylate group, an amino acid, or amixture thereof, with a cross-linker comprising a compound having threeor more functional groups or a mixture of a compound having three ormore functional groups and a compound having two functional groups,

wherein each of the functional groups can react with a carboxylic group(—COOH), carboxylate group (—COO⁻), aldehyde group (—CHO), hydroxyl(—OH), carbonyl group (—CO), sulfone group (—SO₂), amino group (—NH₂) ornitro group (—NO₂), or a mixture thereof.

The present invention further relates to a stable biodegradable, highwater absorbable γ-PGA hydrogel prepared by the above method. Theinventive γ-PGA hydrogel exhibits good biodegradability, up to 5,000times water absorption capacity, and improved firmness, and long-termstability after fully swelling in water or an aqueous medium.

DETAILED DESCRIPTION OF THE INVENTION

In the method of the present invention, (A) a γ-polyglutamic acid(γ-PGA), a γ-polyglutamate, or a mixture thereof, and optionally apolysaccharide containing a carboxylic and/or carboxylate group, anamino acid, or a mixture thereof; and/or (B) a microbial culture brothcontaining a γ-polyglutamic acid (γ-PGA), a γ-polyglutamate, or amixture thereof, and optionally a polysaccharide containing a carboxylicand/or carboxylate group, an amino acid, or a mixture thereof directlyreact with a cross-linker comprising a compound having three or morefunctional groups or a mixture of a compound having three or morefunctional groups and a compound having two functional groups.Preferably, γ-PGA with a molecular weight of more than 100,000 Daltonsis used. The polysaccharide may be selected from, but is not limited to,a mixture of glucose, fructose, galactose, and glucuronic acid, and amixture of rhamnose, glucose, galactose, and glucuronic acid, and apolycarboxylic acid in which hyaluronic acid as the principal component.As for the amino acid, it can be selected from, but is not limited to,polyaspartic acid, polylysine, aspartic acid, lysine and arginine, andmixtures thereof.

No special limitation on the other components of the microbial culturebroth is necessary. All the components that can be used in a culturebroth and that are obvious to persons skilled in the art would besuitable for use in the cultural broth of the present invention. Inother words, the culture broth used in the present invention can beprepared by any methods known to persons skilled in the art. Forexample, JP 1-174397 discloses using a culture broth composed ofL-glutamic acid and peptone to grow Bacillus subtilis and Bacillusnatto, which can produce γ-PGA.

In the present invention, the species of the compound used as thecross-linker do not require any special limitation. Basically, it ispreferred to select a compound having three or more epoxy functionalgroups or a mixture of a compound having three or more epoxy functionalgroups and a compound having two epoxy functional groups, such aspolyglycidyl ether, as the cross-linker used in the present invention.For example, the polyglycidyl ether can be selected from, but is notlimited to, glycerol triglycidyl ether, di- or polyglycerol polyglycidylether and polyoxyethylene sorbitol polyglycidyl ether, and a mixturethereof.

In one embodiment of the present invention, the compound having three ormore functional groups is glycerol triglycidyl ether and the compoundhaving two functional groups is glycerol diglycidyl ether, and a mixtureof them is used as the cross-linker.

The di- or polyglycerol polyglycidyl ether can be a compound of Formula(I):

wherein R is H or

and n is 2 to 8, preferably n is 2 to 4.

The polyoxyethylene sorbitol polyglycidyl ether can be a compound ofFormula (II):

wherein R is H or

and x, y, z, o, p, and q are independently 1 to 3.

For conducting the cross-linking reaction of the present invention, theamount of the cross-linker, on the basis of the total weight of (A) and(B), is normally 0.02 to 20 wt % and preferably 0.25 to 6 wt %. If theamount of the cross-linker is below 0.02 wt %, the high water absorptionrate cannot be achieved because of the insufficient cross-linking.However, if the amount of the cross-linker is greater than 20 wt %, theresulted γ-PGA hydrogels exhibit low water absorbability due toover-cross-linking.

When conducting the aforementioned cross-linking reaction, thecross-linking system is normally maintained at a pH of 3.3 to 8.5, andpreferably 4.0 to 8.5. Furthermore, the reaction temperature is between0° C. and 100° C., and preferably 35° C. and 85° C. Generally, it takesa longer time to complete a reaction conducted at a lower temperature,and on the contrary, a shorter time for the reaction conducted at ahigher temperature. Nonetheless, if the reaction temperature is higherthan 100° C., undesired side reactions, such as decomposition, will takeplace and influence the effectiveness of the cross-linking. In addition,carboxylic group (—COOH), carboxylate group (—COO⁻), aldehyde group(—CHO), hydroxyl (—OH), carbonyl group (—CO), sulfone group (SO₂), aminogroup (—NH₂) or nitro group (—NO₂), or a combination thereof to theepoxy group provided by the cross-linker is 1:1.

In the method of the present invention, the manner for carrying out thecross-linking reaction does not require any special limitation. Forexample, glass reactors equipped with stirrer devices or culturecontainers shaked in an oil or water bath can be utilized to accomplishthe cross-linking reaction involved in the present invention. The methodof the present invention may further comprise the steps of hydrating thecross-linked products for swelling, removing the un-cross-linkedcomponents by filtration, and drying (e.g., lyophilizing) the preparedwater absorbable cross-linked product, to obtain the cross-linkedproduct with high water absorbability.

Apparently, the method of the present invention can produce high waterabsorbable and stable biodegradable γ-PGA hydrogels more simply and moreeasily as compared with conventional methods.

The present invention further relates to a stable biodegradable, highwater absorbable γ-PGA hydrogel prepared by the above method. The γ-PGAhydrogel of the present invention is effective in terms of waterabsorption and retention, provides up to 5,000 times water absorptionrate, and can be decomposed by microbes existing in the naturalenvironment so that its waste treatment is safer and simpler. Mostimportantly, since the cross-linker, which comprises a compound havingthree or more functional groups or a mixture of a compound having threeor more functional groups and a compound having two functional groups,is used to conduct the cross-linking reaction, the γ-PGA hydrogel of thepresent invention has 3-dimensional inter-molecular cross-linked matrixand thus exhibits a longer stability and improved firmness and strengthafter full swelling in water or an aqueous medium without disintegratingor breaking down even for over 5 weeks in open atmosphere at temperatureof 30° C.

The stable biodegradable, high water absorbable γ-PGA hydrogel of thepresent invention can be used in fields including, among others, thecosmetics fields, as moisturizers or humectants, agricultural andhorticultural fields, as soil reconditioning agents, seed-coatingagents, water-retaining agents for plant cultivation, immobilizingagents for manure of animals, compost adding agents, or moisturizers forfeces, urine, etc.; the civil construction field, as water conditioningagents for water treatment sludge, sewage sludge, and river sewersludge, solidifying agents, modifying agents, coagulants, or soil forreservoir; medical and hygiene fields, as absorbents for bloods or bodyfluids, paper diapers or tampons, or as de-odorants or controlledrelease drug carriers; and the bioengineering fields, as medium base forculturing microbes, plant cells or animal cells, or as immobilizingmaterials for bioreactors.

Moreover, since γ-PGA hydrogel of the present invention can be preparedby directly reacting the culture broth (Component (B)) with across-linker, it will inherently contain the components of the culturebroth necessary for the growth of microbes, such as carbon source,nitrogen source, and minerals, and/or metabolites produced by microbesin the culture broth. In view of this property, the biodegradable, waterabsorbable γ-PGA hydrogel of the present invention is very suitable foruse as an agricultural material for compost aids, seed coating agents,and desert greenification materials.

The biodegradable, water absorbable γ-PGA hydrogel of the presentinvention can be in any desired shapes. For example, the hydrogels canbe granulated into a fixed shape or made into irregular shapes, pellets,plates, etc.

The subject invention will be further described by the followingexamples. Nonetheless, it should be noted that the working examples areprovided for an illustration of the present invention, rather thanintended to limit the scope of the present invention.

EXAMPLE 1

A 300 L culture medium containing 0.5 wt % of yeast extract, 1.5 wt % ofpeptone, 0.3 wt % of urea, 0.2 wt % of K₂HPO₄, 10 wt % of L-glutamicacid, and 8 wt % of glucose and having a pH of 6.8 was added to a 600 Lfermentor, and then steam sterilized following the standard procedures.Bacillus subtilis was incubated under 37° C. After 96 hours, the culturebroth contained 40 g γ-PGA per liter. Each of 15 g of the culture brothwas added to 50 ml capped sample bottles into which each of 600 μl ofthe cross-linkers as listed in Table 1 is introduced. The reaction ofthe mixtures were conducted at 55° C. for 20 hours in a shakerincubator, rotating at a middle speed.

Each of 1 g of the reacted mixtures were taken out of the 50 ml cappedsample bottles and soaked in 800 ml of water at 4° C. overnight. Thecross-linked hydrogel formed after hydration and swelling was thenfiltered through an 80-mesh metal screen and drained to dry. The weightsof swollen hydrogels without obvious free water were measured andrecorded. The hydrogels were re-soaked in another 800 ml of fresh waterat 4° C. in the same beaker overnight. The same procedure was repeatedfor consecutive 5 days. The cross-linked product was then tested for itswater absorption rate as follows:

For the determination of water absorption rate, the cross-linked productwas soaked in an excess amount of distilled water and left in the waterfor swelling overnight to achieve highest hydration. An 80-mesh metalscreen was used to filtrate the excess amount of water to obtain thewetted cross-linked product. The wetted cross-linked product wasweighed. The water absorption rate is defined as the ratio of the weightof water absorbed (the difference between the wet and dry weights) tothe dry weight. The results of the water absorption rate for thisexample are shown in Table 1.

TABLE 1 Reaction Water Cross-linker time (hr) absorption ratePolyethylene glycol diglycidyl ether 20 4,500 A mixture of glyceroltriglycidyl ether with 20 4,600 glycerol diglycidyl ether Diglycerolpolyglycidyl ether 20 4,880 Polyglycerol polyglycidyl ether 20 4,950Polyoxyethylene sorbitol polyglycidyl ether 20 4,750polyethylene glycol diglycidyl ether is a mixture comprising compoundshaving the formula of

wherein x is 1, 2, 4, 9, 13 or 22

The results listed in Table 1 show that the utilization of either thecompound having only two epoxy functional groups, or the compoundshaving three or more epoxy functional groups or a mixture of a compoundhaving three or more epoxy functional groups and a compound having twoepoxy functional groups as the cross-linker can attain at least 4,500times water absorption rate.

EXAMPLE 2

According to the procedures illustrated in Example 1, samples of 5 wt %sodium γ-PGA solutions and a mixture of glycerol triglycidyl ether withglycerol diglycidyl ether as the cross-linker were used in another setof experiments. The pH was varied as those listed in Table 2. Thereacted mixtures were further put into an culture shaker, rotating at amiddle speed. The reaction was allowed to continues at 55° C. for 20hours. After the reaction was completed, the water absorption rates weredetermined. The results of the water absorption rates of the obtainedcross-linked products are listed in Table 2.

TABLE 2 pH Water absorption rate 4.0 435 5.0 610 6.0 3,450 8.0 4,550

EXAMPLE 3

According to the procedures illustrated in Example 1, samples of 5 wt %sodium γ-PGA solutions and diglycerol polyglycidyl ether as thecross-linker were used in another set of experiments. The pH of thesolutions was adjusted to 6.5. The amounts of diglycerol polyglycidylether listed in Table 3 were used for the cross-linking reaction. Thereaction was allowed to continue at 55° C. for 20 hours. The results ofwater absorption rates of the obtained cross-linked products are listedin Table 3.

TABLE 3 Amount of Water absorption rate cross-linker Swelling/hydrationtime (hr) (%) 24 48 72 96 120 2 450 1,250 2,550 4,350 4,450 3 359 1,0032,200 4,200 4,480 4 — — 2,090 4,050 4,350

EXAMPLE 4

According to the procedures illustrated in Example 1, Bacillus subtiliswas incubated. The incubation time was altered as shown in Table 4. ThepH of the solutions was adjusted to 6.5. A mixture of glyceroltriglycidyl ether with glycerol diglycidyl ether was used as thecross-linker. Then, the obtained culture broth was used to conductcross-linking at 55° C. for 20 hours. The results of the waterabsorption rates of the obtained cross-linked products are shown inTable 4.

TABLE 4 Incubation time (hr) Water absorption rate 24 a) 36 a) 48 2,60060 3,050 72 3,000 84 2,880 96 3,550 a): No cross-linked hydrogel isformed.

EXAMPLE 5

According to the procedures illustrated in Example 1, samples of theculture broth at 96^(th) hour (3.8 wt % of γ-PGA) and samples of 3.8 wt% of γ-PGA solutions prepared from purified sodium γ-polyglutamate wereused to separately react with diethylene glycol diglycidyl ether andwith polyglycerol polyglycidyl ether. The pH of the solutions wasadjusted to 6.5. Both of the epoxy compounds were used at 3 wt % of theculture broth or the γ-PGA solution. The cross-linking reactions wereallowed to continue at 55° C. for 20 hours. The resulted hydrogelsamples were hydrated in excess amount of water at 4° C. for 24 hours.Then, the samples were again hydrated in another fresh water. The sameprocedure was repeated for 3 consecutive days. The fully swollenhydrogel samples in water were kept at 30° C. in open atmosphere forobserving the physical stability and integrity over a period ofconsecutive 35 days. The soaking water of each hydrogel sample wasreplaced every 24 hours. The results of the physical stability andintegrity of the swollen hydrogel samples were listed in Table 5.

TABLE 5 Stability and integrity Condition observed after samples wereCross-linker placed at 30° C. in open atmosphere (3 wt %) 3 days 5 days35 days Purified γ-PGA with Partially Completely — diethylene glycoldecomposed decomposed diglycidyl ether Purified γ-PGA with Good and Goodand Good and polyglycerol firm firm firm polyglycidyl ether Culturebroth with Partially Completely — diethylene glycol decomposeddecomposed diglycidyl ether Culture broth with Good and Good and Goodand polyglycerol firm firm firm polyglycidyl ether

The results in Table 5 show that the utilization of a compound havingthree or more epoxy functional groups, as the cross-linker can prepare aγ-PGA hydrogel with a high water adsorption rate, long stability afterswelling in water, and good biodegradability.

1. A biodegradable, water absorbable γ-polyglutamic acid hydrogel whichis prepared by directly cross-linking (A) a γ-polyglutamic acid (γ-PGA),a γ-polyglutamate, or a mixture thereof, and optionally a polysaccharidecontaining a carboxylic group and/or carboxylate group, an amino acid,or a mixture thereof; or (B) a microbial culture broth containing aγ-polyglutamic acid (γ-PGA), a γ-polyglutamate, or a mixture thereof,and optionally a polysaccharide containing a carboxylic group and/orcarboxylate group, an amino acid, or a mixture thereof; a cross-linkercomprising a polyglycidyl ether having three or more functional groups,wherein the functional groups thereon can react with a carboxylic group(—COOH), carboxylate group (—COO⁻), aldehyde group (—CHO), hydroxyl(—OH), carbonyl group (—CO), sulfone group (—SO₂), amino group (—NH₂) ornitro group (—NO₂), or a mixture thereof; and wherein said polyglycidylether is glycerol triglycidyl ether, di- or polyglycerol polyglycidylether and polyoxyethylene sorbitol polyglycidyl ether, or a mixturethereof.
 2. The hydrogel of claim 1, wherein the polysaccharide isselected from the group consisting of a mixture of glucose, fructose,galactose, and glucuronic acid, and a mixture of rhamnose, glucose,galactose, and glucuronic acid, and a polycarboxylic acid in whichhyaluronic acid is the principal component.
 3. The hydrogel of claim 1,wherein the amino acid is selected from the group consisting ofpolyaspartic acid, polylysine, aspartic acid, lysine and arginine, and amixture thereof.
 4. The hydrogel of claim 1, wherein the polyglycidylether is glycerol triglycidyl ether.
 5. The hydrogel of claim 1, whereinthe di- or polyglycerol polyglycidyl ether is a compound of Formula (I):

wherein R is H or

and n is 2 to
 8. 6. The hydrogel of claim 5, wherein n is 2 to
 4. 7. Thehydrogel of claim 1, wherein the polyoxyethylene sorbitol polyglycidylether is a compound of Formula (II):

wherein R is H or

and x, y, z, o, p, and q are independently 1 to
 3. 8. The hydrogel ofclaim 1, wherein the amount of the cross-linker is 0.02 to 20 wt %,based on the total weight of (A) and (B).
 9. The hydrogel of claim 8,wherein the amount of the cross-linker is 0.25 to 6 wt %, based on thetotal weight of (A) and (B).
 10. The hydrogel of claim 1, wherein thecross-linking is conducted at a temperature of from 0° C. to 100° C. 11.The hydrogel of claim 10, wherein the cross-linking is conducted at atemperature of from 35° C. to 85° C.
 12. The hydrogel of claim 1,wherein the cross-linking is conducted at a pH of from 3.3 to 8.5. 13.The hydrogel of claim 12, wherein the cross-linking is conducted at a pHof from 4.0 to 8.5.
 14. The biodegradable, water absorbableγ-polyglutarnic acid hydrogel of claim 1 has 3-dimensional stabilityafter swelling in water or an aqueous medium.
 15. The hydrogel of claim14 which is a long-lasting hydrogel without disintegrating or breakingdown for over 5 weeks in atmosphere at temperature of 30° C.
 16. Thehydrogel of claim 15 comprising the components of culture brothnecessary for the growth of microbes and/or metabolites produced bydirectly crosslinking.